For a number of reasons including ease of cell culture, genetic manipulation and experimental design the social amoeba Dictyostelium discoideum has long been a model system for investigating the physiological roles of the actomyosin cytoskeleton. One of our interests in recent years has been the functions of the actin cross-linking proteins cortexillins (ctx) I, II and III. We had shown that the double knockout of ctxI and ctxII completely inhibits, cell streaming and development by decreasing the secretion of cAMP. More recently, we have focused on ctxIII. Unlike recombinant cortexillins I and II, which form both homodimers and heterodimers, we find that cortexillin III expressed in E. coli forms unstable monomers but not homodimers, and significantly stable heterodimers when coexpressed with either cortexillin I or II. None of the recombinant cortexillin homo- or hetero-dimers affects actin polymerization in vitro and only the cortexillin II homodimers bind to F-actin with high affinity. Cortexillin III expressed in Dictyostelium wild-type or cortexillin III-null cells forms heterodimers with both cortexillin I and II and binds to DGAP1 (a Dictyostelium GAP protein) but only as a heterodimer, i.e. expressed cortexillin III does not bind to DGAP1 in cortexillin I and II double-null cells. Heterodimers of expressed cortexillin III colocalize with F-actin in the cortex of vegetative cells, the leading edge of motile cells and the cleavage furrow of dividing cells but not in DGAP1-null cells; i.e. cortexillin III, and possibly also cortexillins I and II, binds to F-actin in vivo only when complexed to DGAP1. Cortexillin III-null cells chemotax normally but in the developmental cycle the null-cells form unstable streams and smaller mounds and smaller fruiting bodies than wild-type cells. Cortexillin III may be a negative regulator of pinocytosis, phagocytosis, cytokinesis and cell growth as all are enhanced in cortexillin III-null cells.